Iberiotoxin block of endotoxin tolerance in a mouse macrophage cell line.

Minae Yoshida, Alasdair Gibb, Dean Willis. University College London, London, UK

RAW264.7, a mouse macrophage cell line, is a standard model for investigating cell signalling mechanisms during macrophage activation. Leukocytes are known to be electrogenic (DeCoursey, 2007) and macrophages express an array of ion channels (Gallin, 1991). The role, however, of individual ion channel types in RAW264.7 activation is not fully understood. In this study we focused on the role of BK channels in the activation of RAW264.7 cells by the toll-like receptor 4 agonist, LPS and endotoxin tolerance. Using both pharmacological and electrophysiological techniques we present evidence for a role for BK channels regulating endotoxin tolerance.

Cytokine release from RAW264.7 cells in response to varying concentration of LPS (1, 10, 30, 100, 300, 1000 ng/ml) was measured by ELISA (BD Biosciences BD OptEIA kits for TNFα, IL-6 and IL-10) 4 hours after stimulation. Here, for brevity, we present the results for TNFα. To evaluate the role of BK channels in RAW264.7 cell activation we used the selective inhibitor, iberiotoxin (1, 3, 10, 30 nM) (Gribkoff, 1995). To elicit endotoxin tolerant cells, we pre-stimulated cells with low dose LPS (10 ng/ml) for 24 hours prior to the second high dose LPS (150 ng/ml) challenge. Cytokine production in response to the second high LPS dose challenge was measured after 4 hours. Iberiotoxin (10 nM) was applied to the cells either during the low dose pre-treatment or the high dose challenge. Stock Iberiotoxin solution was made up in 0.9% saline and LPS stock solution in distilled water. These solutions were then diluted in DMEM culture media to the required concentration. The control groups recieved DMEM as vehicle. Expression of BK channels in stimulated or non-stimulated RAW264.7 cells at various time points was assessed using a voltage-ramp protocol in whole-cell patch-clamp recordings where the pipette solution contained either low (˜20 nM) or high (˜640 nM) EGTA-buffered free calcium concentration. Experiments were repeated at least 3 times, the results are expressed as mean +/-SEM and statistical significance tested using the Student’s t-test.

TNFα secretion could not be detected from control or Iberiotoxin (10 nM) treated RAW 264.7 cells after 4 or 24 hours (TNFα <30 pg/ml: detection limit of assay). LPS dose-dependently increased the TNFα production from RAW cells (EC50 = 13.5 ng/ml). This was unaltered by iberiotoxin. Pre-stimulation of cell with low dose LPS significantly decreased TNFα production in response to a second high dose LPS challenge (TNFα 2.44+/-0.37 ng/ml) compared to the groups with no pre-stimulation (3.77+/-0.22 ng/ml, P=0.011, n=6). Iberiotoxin treatment (10 nM) reversed this endotoxin tolerance. This was seen when the drug was applied during the low-dose LPS pre-stimulation period (3.89 +/-0.13 ng/ml, P=0.014, n=6) or the second high dose challenge (TNFα 3.97+/-0.32 ng/ml, n=6, P=0.016) Taken together, these results suggest that BK channels may have a role in regulating the macrophage response to prolonged LPS activation and maintenance of endotoxin tolerance.

References:

Decoursey T (2007) American Journal of Physiology Cell Physiology 293:C30-32.

Gallin E (1991) Physiological Reviews, 71:775-811.

Gribkoff V et al (1995) Molecular Pharmacology 50:206-217.